Proteins play an important role in today's medical portfolio. For human applications every therapeutic protein has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans, it is especially important that by-products which accumulate during production processes be removed. To fulfill regulatory specifications, one or more purification steps have to follow the manufacturing process. Among other things, purity, throughput, and yield play important roles in determining an appropriate purification process.
Different methods are well established and their use for protein purification is widespread, such as affinity chromatography with microbial proteins (e.g., protein A and protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (sulfopropyl or carboxymethyl resins), anion exchange (amino ethyl resins and mixed-mode ion exchange), thiophilic adsorption (e.g., with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, and capillary electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102)).
Conjugates of polyethylene glycol (PEG) and, e.g., Interleukin-6 (EP 0 442 724), erythropoietin (WO 01/02017), chimeric molecules comprising Endostatin and immunoglobulins (US 2005/008649), secreted antibody based fusion proteins (US 2002/147311), fusion polypeptides comprising albumin (US 2005/0100991, human serum albumin U.S. Pat. No. 5,876,969), PEGylated polypeptides (US 2005/0114037), and for interferon fusions have been reported.
Necina, R., et al. (Biotechnol. Bioeng. 60 (1998) 689-698) reported the capture of human monoclonal antibodies directly from cell culture supernatants by ion exchange media exhibiting high charge density. In WO 89/05157 a method is reported for the purification of immunoglobulins by directly subjecting the cell culture medium to cation exchange treatment. One-step purification of monoclonal IgG antibodies from mouse ascites is described by Danielsson, A., et al., J. Immun. Meth. 115 (1988), 79-88. A method for purifying a polypeptide by ion exchange chromatography is reported in WO 2004/024866 in which a gradient wash is used to resolve a polypeptide of interest from one or more contaminants. In EP 0 530 447 a process for purifying IgG monoclonal antibodies by a combination of three chromatographic steps is reported. A facile purification of mono-PEGylated interleukin-1 receptor antagonist is reported by Yu, G., et al., in Process Biotechnol. 42 (2007) 971-977. Wang et al. (Wang, H., et al., Peptides 26 (2005) 1213-1218) reports the purification of hTFF3 expressed in E. coli by two step cation exchange chromatography. Yun et al. (Yun, Q., et al., J. Biotechnol. 118 (2005) 67-74) report the purification of PEGylated rhG-CSF by two consecutive ion-exchange chromatography steps. WO 2007/039436 and WO 01/087329 report erythropoietin being covalently linked to poly (ethylene glycol) group(s) and a liquid pharmaceutical composition comprising an erythropoietin protein.